CN117448072A - Olive leaf polyphenol extract, polyphenol refined olive oil and preparation method thereof - Google Patents

Abstract

The invention provides an olive leaf polyphenol extract, polyphenol refined olive oil and a preparation method thereof. The present invention relies on dark standing treatment, microwave-assisted extraction, and degreening treatment to obtain an olive leaf polyphenol extract that removes chlorophyll, and then enriches the olive leaf polyphenol extract into refined olive oil to prepare a high-containing polyphenol extract. The refined olive oil of the substance simultaneously solves the technical problems of low oil yield and high chlorophyll content.

Description

Olive leaf polyphenol extract, polyphenol refined olive oil and preparation method thereof

Technical field

The invention relates to the technical field of olive oil preparation, and specifically relates to an olive leaf polyphenol extract, polyphenol refined olive oil and a preparation method thereof.

Background technique

The European Food Safety Authority (EFSA) issued a health statement on all commercially available olive oil in 2011 and pointed out that olive oil containing at least 250 mg of polyphenols per 1 kg of oil is an olive oil that meets health requirements. The content of polyphenols in conventionally consumed refined olive oil is extremely low, in most cases less than 40 mg/kg, which is far less than the content required by the EFSA health statement.

At present, technical solutions to specifically improve the polyphenols in refined olive oil have problems such as a significant decrease in oil yield or a deepening of color due to high leaf green content.

Contents of the invention

Based on this, there is a need to provide a refined olive oil containing high polyphenols and a preparation method thereof.

The present invention adopts the following technical solutions:

The invention provides a polyphenol refined olive oil with a total phenolic content greater than 250 mg/kg and a chlorophyll content less than 15 mg/kg. The polyphenol refined olive oil also contains hydroxytyrosol, mignonette glycerin, quercetin and Oleacein. .

The invention provides a method for preparing polyphenol refined olive oil, which includes the following steps:

Get olive leaf polyphenol extract powder;

The olive leaf polyphenol extract and refined olive oil are evenly mixed according to a mass ratio of 1:15 to 1:40, subjected to high-pressure dissolution-enhancing enrichment and/or pulse ultrasonic dissolution-enhancing enrichment treatment, and centrifugal filtration to obtain a high-content polyphenol extract. Phenolics in refined olive oil.

In some of the embodiments, preferably, the process condition parameters of the high-pressure solution-promoting treatment are: pressure 150 to 250 MPa, and treatment time 3 to 10 minutes.

In some of the embodiments, preferably, the process condition parameters of the pulsed ultrasonic solubilization treatment are: ultrasonic power is 450-650W, total ultrasonic treatment time is 2-10 minutes, ultrasonic pulse interval program is 10s on, 5s off, ultrasonic Frequency 250~350Hz, water bath temperature 30~45℃.

In some of the embodiments, preferably, the process condition parameters of centrifugal filtration are: first use a disc centrifuge at 3500g for 10 minutes to remove solid particles, then let it stand in the dark and then filter with an industrial-grade oil filter.

In some embodiments, the preparation method steps of the olive leaf polyphenol extract powder are:

S1, obtain olive leaf raw materials, clean them, and obtain material A;

S2, mix material A with water, add acid to adjust the pH to 5.5-6.5, carry out the reaction in the dark under stirring conditions and avoid light, add alkali solution to adjust the pH to 6.5-7.0, carry out the reaction in the dark, and dry in a vacuum , crushed to obtain reaction material B;

S3, mix the reaction material B with the extraction solvent, and perform microwave-assisted extraction to obtain extract liquid C;

S4, add acid to the extract C to adjust the pH to 2.5-3.5, mix evenly, add alkali solution dropwise to adjust the pH to 9.5-10.0, let it stand at low temperature to precipitate chlorophyll, and filter to obtain the extract D;

S5, mix the extraction solution D with the 40% ethanol aqueous solution, let it stand for layering, and collect the ethanol aqueous phase extract E;

S6: Distill the aqueous ethanol extract E to obtain the ethanol-removed product, which is then freeze-dried at low temperature to obtain olive leaf extract powder.

Preferably, in step S1, the olive leaf raw material is selected from late spring young leaves of olives of four varieties: Piguar, Collati, Berat, and Godard.

Preferably, in step S2, material A and water are mixed according to the material-liquid ratio of 1g:30mL~1g:70mL, add acid to adjust the pH to 5.5~6.5, and stir under the conditions of 35~45°C and 45~50% humidity. Carry out the reaction in the dark and avoid light for 20 to 24 hours, add alkali solution to adjust the pH value to 6.5 to 7.0, let it stand for another 20 to 24 hours in the dark, vacuum dry at 35 to 45°C, and pulverize to obtain reaction material B.

Preferably, in step S3, the extraction solvent is an aqueous solution containing choline chloride and malic acid in a mass ratio of 1:2 to 1:4. The mass ratio of reaction material B to extraction solvent is 1:10~1:14. The process conditions for microwave-assisted extraction are: microwave power 600~700W, microwave treatment time 15~30min, and treatment temperature 40~50°C.

Preferably, in step S4, the condition parameter of low-temperature standing is: standing at -20~-5°C for 60~120 minutes.

Preferably, in step S5, the ethanol aqueous solution is 20% to 55% ethanol aqueous solution, and the volume ratio of the extraction liquid D to the ethanol aqueous solution is 1:1 to 1:4.

Preferably, in step S6, the process parameters of distillation are: steam pressure 0.2~2.0Mpa, distill until there is no ethanol in the solution and collect the absolute ethanol obtained by distillation for recycling, retain the aqueous solution and the precipitated precipitation, and put them together - Store at 20~-5℃ for 8~12h. The process parameters of low-temperature freeze-drying are: temperature -40~-30℃, vacuum degree 20~100Pa, and processing time 20~24h.

Compared with the prior art, the beneficial effects of the present invention are:

The invention provides an olive leaf polyphenol extract and a method for preparing polyphenol refined olive oil containing the olive leaf polyphenol extract. The prepared olive leaf polyphenol extract is enriched into the refined olive oil, so that the polyphenols are The content of similar substances is increased to no less than 250mg/kg, while the chlorophyll content is significantly reduced, which can also overcome the problem of reduced oil yield.

Description of the drawings

Figure 1 is an HPLC chromatogram of four main polyphenolic substances in the polyphenol refined olive oil prepared in Example 4.

Figure 2 shows the HPLC chromatograms of polyphenol refined olive oil prepared in different test examples.

Figure 3 is a comparison of the appearance and color effects of polyphenol refined olive oil prepared in Example 2 and Example 4 and commercial olive oil (respectively referred to as "refined oil" and "commercial extra virgin olive oil").

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments to enable those skilled in the art to understand the present invention more clearly. The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless otherwise specified, the technical means used are all commercially available products familiar to those skilled in the art. well-known conventional means.

Description of sources of some key test raw materials:

Refined olive oil: Use the Monini brand of refined olive oil from Italy purchased through online platforms.

Raw materials for olive leaves: Collect 1.0kg each of four varieties of olive leaves in late spring (March) including Piguar, Corati, Berat, and Godard, mix them evenly in the same proportion, and screen out the dark green old leaves. Keep the younger leaves, which are lighter in color. The interval between olive leaves being picked from the tree should not exceed 48 hours to ensure that the endogenous enzymes of the olive leaves are still maintained at a high level of enzyme activity.

Example 1

This embodiment provides a method for preparing polyphenol refined olive oil, which includes the following steps:

S1, olive leaf screening and cleaning:

Use fans and vibrating screens to separate and screen out the small branches mixed in the olive leaf raw materials. The olive leaf raw materials mixed in equal proportions are washed multiple times to remove dust and insect eggs adhered to the leaves to obtain clean olive leaves A. Note: The interval between olive leaves A and being picked from the tree should not exceed 48 hours to ensure that the endogenous enzymes of the olive leaves are still maintained at a high level of enzyme activity.

S2, dark static preprocessing:

Mix fresh and clean olive leaves A with pure water into a high-speed homogenizer mixer. The material-to-liquid ratio is 1:30 (g/mL). Beat at a constant speed of 50 rpm. Add a small amount of acetic acid to adjust the pH of the solution to 6.0. The temperature Control to 40°C and ambient air humidity of 50% for 24 hours in the dark and light-proof conditions. Turn off the mixer, add an appropriate amount of sodium bicarbonate to adjust the pH to 7.0, and let the solution stand in the dark for another 24 hours. Use a vacuum drying device to dry the solution at 40°C, collect all dry solids, crush it using a pulverizer, and pass it through a 100-mesh sieve to obtain olive leaf powder B (referred to as "reaction material B").

S3, deep eutectic solvent coupled microwave extraction of olive leaf polyphenols:

Preparation of deep eutectic solvent (extraction reagent): Mix choline chloride and malic acid at a mass ratio of 1:2, stir rapidly at 200 rpm in an 80°C water bath, and form a viscous clear liquid after 5 to 10 minutes. Add 1.5 times the volume of water and mix thoroughly to obtain the deep eutectic solvent required for extraction.

Mix the olive leaf powder B and the prepared deep eutectic solvent at a mass ratio of 1:10, and use microwave-assisted extraction: microwave power 700W, microwave treatment time 15min, and treatment temperature 50°C. After treatment, use a self-cleaning filter to obtain olive leaf polyphenol DES extract A (which can also be referred to as "extract C").

S4, degreening treatment of olive leaf polyphenol extract:

Add glacial acetic acid to olive leaf polyphenol DES extract A, adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w/v) sodium bicarbonate solution, adjust the pH to about 9.5, and let stand at -20°C. After 2 hours, chlorophyll precipitated under the control of pH and temperature, and olive leaf polyphenols remained in the DES extraction solution. Take the filtered DES extract (abbreviated as "Extract D") and mix it with 40% ethanol solution at a volume ratio of 1:1, stir thoroughly and mix well, let it stand for layering, and take the upper 40% ethanol aqueous phase to obtain the removal Clarified olive leaf polyphenol extract of chlorophyll B (referred to as "extract E").

S5, freeze drying process into powder:

Use olive leaf polyphenol extract B to separate ethanol and water using distillation equipment. The steam pressure is 0.2Mpa. Distill until there is no ethanol in the solution and collect the distilled absolute ethanol for recycling. Keep the aqueous solution and the precipitated precipitation and put them together. Store at -20℃ for 12h. The frozen block aqueous solution and the precipitated precipitate are freeze-dried to obtain dried olive leaf enriched powder A.

Among them, the freeze-drying process conditions are temperature -40°C, vacuum degree 20Pa, and processing time 24 hours. The olive leaf enriched powder A is further pulverized using an olive leaf vibrating ultra-fine pulverizer, and the olive leaf enriched powder B is filtered with a vibration screening machine of 400 mesh to obtain the olive leaf enriched powder B.

S6, ultrasonic pulse enrichment processing:

Mix olive leaf enriched powder B and refined olive oil (commercially available, also referred to as "refined oil") at a mass ratio of 1:10. Use an ultrasonic pulse crusher to process the enriched powder and oil. The process parameters are as follows: The ultrasonic power is 600W, the ultrasonic treatment time is 3min (ultrasonic pulse interval time, 5s on, 5s off), the ultrasonic frequency is 300Hz, and the water bath temperature is 35°C. After ultrasonic crushing treatment, enriched refined olive oil A is obtained.

S7, separation and filtering:

The enriched refined olive oil A is separated using a disc centrifuge (centrifugal acceleration 3500g, separation time 10 minutes) to obtain solid particles and clarified olive oil B. The clarified olive oil B is in the upper layer, and the solid particles are precipitated in the lower layer. The clarified olive oil B was left to stand for 24 hours under dark conditions, and filtered using an industrial-grade oil filter to obtain the final clear, impurity-free polyphenol-enriched olive oil C.

Example 2

This embodiment provides a method for preparing polyphenol refined olive oil, which includes the following steps:

The method steps from S1 to S3 to prepare olive leaf polyphenol DES extract A (which may also be referred to as “extract C”) are the same as those in Example 1.

S4, extract olive leaf polyphenol extract (without degreening):

Mix olive leaf polyphenol DES extract A (abbreviated as "extract C") directly with 40% ethanol solution at a volume ratio of 1:1, stir thoroughly, let stand and separate into layers, take the upper 40% ethanol aqueous phase, A clear olive leaf polyphenol extract B (referred to as "extract E") was obtained.

S5, freeze drying process into powder.

This operation step is the same as that in Example 1.

S6, ultra-high voltage coupled ultrasonic pulse enrichment processing:

Mix the olive leaf enriched powder B and refined olive oil evenly at a mass ratio of 1:10, and use ultra-high pressure technology to dissolve the active ingredients in the olive leaf enriched powder B into the oil. The pressure is 200MPa and the processing time is 3 minutes. Then use an ultrasonic pulse crusher to process the enriched powder and oil. The process parameters are as follows: ultrasonic power is 600W, ultrasonic treatment time is 3 minutes (ultrasonic pulse interval, working 5s, stopping 5s), ultrasonic frequency 300Hz, water bath temperature 35°C. After ultrasonic crushing treatment, enriched refined olive oil A is obtained.

S7, separation and filtering:

The enriched refined olive oil A is separated using a disc centrifuge (centrifugal acceleration 3500g, separation time 10 minutes) to obtain solid particles and clarified olive oil B. The clarified olive oil B is in the upper layer, and the solid particles are precipitated in the lower layer. The clarified olive oil B was left to stand for 24 hours under dark conditions, and filtered using an industrial-grade oil filter to obtain the final clear, impurity-free polyphenol-enriched olive oil C.

Example 3

This embodiment provides a method for preparing polyphenol refined olive oil, which includes the following steps:

S1, olive leaf screening and cleaning:

This operation step is the same as that in Example 1.

S2, dark static preprocessing:

This operation step is the same as that in Example 1.

S3, microwave extraction of olive leaf polyphenols:

Mix the olive leaf powder B pretreated by standing in the dark with 80% ethanol aqueous solution (the extraction reagent does not contain choline chloride and malic acid) in a mass ratio of 1:10, microwave-assisted extraction, microwave power 700W, microwave treatment The time is 15 minutes and the treatment temperature is 50°C. After treatment, a self-cleaning filter was used to obtain olive leaf polyphenol ethanol extract A.

S4, degreening treatment of olive leaf polyphenol extract:

This operation step is the same as that in Example 1.

S5, freeze drying process into powder:

This operation step is the same as that in Example 1.

S6, ultra-high voltage coupled ultrasonic pulse enrichment processing:

This operation step is the same as that in Example 2.

S7, separation and filtering:

This operation step is the same as that in Example 2.

Example 4

This embodiment provides a method for preparing polyphenol refined olive oil, which includes the following steps:

S1, olive leaf screening and cleaning:

The olive leaf raw materials mixed in equal proportions are washed multiple times to remove dust and insect eggs adhered to the leaves to obtain clean olive leaves A. Note: The interval between olive leaves A and being picked from the tree should not exceed 48 hours to ensure that the endogenous enzymes of the olive leaves are still maintained at a high level of enzyme activity.

S2, dark static preprocessing:

Mix fresh and clean olive leaves A with pure water into a high-speed homogenizer mixer. The material-to-liquid ratio is 1:30 (g/mL). Beat at a constant speed of 50 rpm. Add a small amount of acetic acid to adjust the pH of the solution to 6.0. The temperature Control to 40°C and ambient air humidity of 50% for 24 hours in the dark and light-proof conditions. Turn off the mixer, add an appropriate amount of sodium bicarbonate to adjust the pH to 7.0, and let the solution stand in the dark for another 24 hours. Use a vacuum drying device to dry the solution at 40°C, collect all dry solids, crush it using a pulverizer, and pass it through a 100-mesh sieve to obtain olive leaf powder B (referred to as "reaction material B").

S3, deep eutectic solvent coupled microwave extraction of olive leaf polyphenols:

Preparation of deep eutectic solvent (extraction reagent): Mix choline chloride and malic acid at a mass ratio of 1:2, stir rapidly at 200 rpm in an 80°C water bath, and form a viscous clear liquid after 5 to 10 minutes. Add 1.5 times the volume of water and mix thoroughly to obtain the deep eutectic solvent required for extraction.

Mix the olive leaf powder B and the prepared deep eutectic solvent at a mass ratio of 1:10, and use microwave-assisted extraction: microwave power 700W, microwave treatment time 15min, and treatment temperature 50°C. After treatment, use a self-cleaning filter to obtain olive leaf polyphenol DES extract A (which can also be referred to as "extract C").

S4, degreening treatment of olive leaf polyphenol extract:

Add glacial acetic acid to olive leaf polyphenol DES extract A, adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w/v) sodium bicarbonate solution, adjust the pH to about 9.5, and let stand at -20°C. After 2 hours, chlorophyll precipitated under the control of pH and temperature, and olive leaf polyphenols remained in the DES extraction solution. Take the filtered DES extract (abbreviated as "Extract D") and mix it with 40% ethanol solution at a volume ratio of 1:1, stir thoroughly and mix well, let stand and separate into layers, take the upper 40% ethanol aqueous phase to obtain the removal Clarified olive leaf polyphenol extract of chlorophyll B (referred to as "extract E").

S5, freeze drying process into powder:

Use olive leaf polyphenol extract B to separate ethanol and water using distillation equipment. The steam pressure is 0.2Mpa. Distill until there is no ethanol in the solution and collect the distilled absolute ethanol for recycling. Keep the aqueous solution and the precipitated precipitation and put them together. Store at -20℃ for 12h. The frozen block aqueous solution and the precipitated precipitate are freeze-dried to obtain dried olive leaf enriched powder A.

Among them, the freeze-drying process conditions are temperature -40°C, vacuum degree 20Pa, and processing time 24 hours. The olive leaf enriched powder A is further pulverized using an olive leaf vibrating ultra-fine pulverizer, and the olive leaf enriched powder B is filtered with a vibration screening machine of 400 mesh to obtain the olive leaf enriched powder B.

S6, ultra-high voltage coupled ultrasonic pulse enrichment processing:

Mix the olive leaf enriched powder B and refined olive oil evenly at a mass ratio of 1:10, and use ultra-high pressure technology to dissolve the active ingredients in the olive leaf enriched powder B into the oil. The pressure is 200MPa and the processing time is 3 minutes. Then use an ultrasonic pulse crusher to process the enriched powder and oil. The process parameters are as follows: ultrasonic power is 600W, ultrasonic treatment time is 3 minutes (ultrasonic pulse interval, working 5s, stopping 5s), ultrasonic frequency 300Hz, water bath temperature 35°C. After ultrasonic crushing treatment, enriched refined olive oil A is obtained.

S7, separation and filtering:

The enriched refined olive oil A is separated using a disc centrifuge (centrifugal acceleration 3500g, separation time 10 minutes) to obtain solid particles and clarified olive oil B. The clarified olive oil B is in the upper layer, and the solid particles are precipitated in the lower layer. The clarified olive oil B was left to stand for 24 hours under dark conditions, and filtered using an industrial-grade oil filter to obtain the final clear, impurity-free polyphenol-enriched olive oil C.

Example 5

This embodiment provides a method for preparing polyphenol refined olive oil, which includes the following steps:

S1, olive leaf screening and cleaning:

The olive leaf raw materials mixed in equal proportions were washed several times to remove dust and insect eggs adhered to the leaves to obtain clean olive leaves A. Note: The interval between olive leaves A and being picked from the tree should not exceed 48 hours to ensure that the endogenous enzymes of the olive leaves are still maintained at a high level of enzyme activity.

S2, crushing treatment (replacing the dark standing pretreatment in Example 4):

Crush fresh and clean olive leaves A with a grinder and pass through a 100-mesh sieve to obtain olive leaf powder B (referred to as "reaction material B").

S3, deep eutectic solvent coupled microwave extraction of olive leaf polyphenols:

Preparation of deep eutectic solvent (extraction reagent): Mix choline chloride and malic acid at a mass ratio of 1:2, stir rapidly at 200 rpm in an 80°C water bath, and form a viscous clear liquid after 5 to 10 minutes. Add 1.5 times the volume of water and mix thoroughly to obtain the deep eutectic solvent required for extraction.

Mix the olive leaf powder B and the prepared deep eutectic solvent at a mass ratio of 1:10, and use microwave-assisted extraction: microwave power 700W, microwave treatment time 15min, and treatment temperature 50°C. After treatment, use a self-cleaning filter to obtain olive leaf polyphenol DES extract A (which can also be referred to as "extract C").

S4, degreening treatment of olive leaf polyphenol extract:

Add glacial acetic acid to olive leaf polyphenol DES extract A, adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w/v) sodium bicarbonate solution, adjust the pH to about 9.5, and let stand at -20°C. After 2 hours, chlorophyll precipitated under the control of pH and temperature, and olive leaf polyphenols remained in the DES extraction solution. Take the filtered DES extract (abbreviated as "Extract D") and mix it with 40% ethanol solution at a volume ratio of 1:1, stir thoroughly and mix well, let stand and separate into layers, take the upper 40% ethanol aqueous phase to obtain the removal Clarified olive leaf polyphenol extract of chlorophyll B (referred to as "extract E").

S5, freeze drying process into powder:

Use olive leaf polyphenol extract B to separate ethanol and water using distillation equipment. The steam pressure is 0.2Mpa. Distill until there is no ethanol in the solution and collect the absolute ethanol obtained by distillation for recycling. Keep the aqueous solution and the precipitated precipitation and put them together. Store at -20℃ for 12h. The frozen block aqueous solution and the precipitated precipitate are freeze-dried to obtain dried olive leaf enriched powder A.

Among them, the freeze-drying process conditions are temperature -40°C, vacuum degree 20Pa, and processing time 24 hours. The olive leaf enriched powder A is further pulverized using an olive leaf vibrating ultra-fine pulverizer, and the olive leaf enriched powder B is filtered with a vibration screening machine of 400 mesh to obtain the olive leaf enriched powder B.

S6, ultra-high voltage coupled ultrasonic pulse enrichment processing:

Mix the olive leaf enriched powder B and refined olive oil evenly at a mass ratio of 1:10, and use ultra-high pressure technology to dissolve the active ingredients in the olive leaf enriched powder B into the oil. The pressure is 200MPa and the processing time is 3 minutes. Then use an ultrasonic pulse crusher to process the enriched powder and oil. The process parameters are as follows: ultrasonic power is 600W, ultrasonic treatment time is 3 minutes (ultrasonic pulse interval, working 5s, stopping 5s), ultrasonic frequency 300Hz, water bath temperature 35°C. After ultrasonic crushing treatment, enriched refined olive oil A is obtained.

S7, separation and filtering:

The enriched refined olive oil A is separated using a disc centrifuge (centrifugal acceleration 3500g, separation time 10 minutes) to obtain solid particles and clarified olive oil B. The clarified olive oil B is in the upper layer, and the solid particles are precipitated in the lower layer. The clarified olive oil B was left to stand for 24 hours under dark conditions, and filtered using an industrial-grade oil filter to obtain the final clear, impurity-free polyphenol-enriched olive oil C.

Using commercially available refined olive oil and commercially available extra virgin olive oil as controls, the polyphenol-enriched olive oil C prepared in the above experimental examples was tested for ingredients. The test indicators and methods include:

1) Determination of total polyphenol content of olive oil:

Accurately weigh 2.0g of olive oil into a 10mL screw cap test tube. Add 1.0 mL of internal standard solution (0.015 mg/mL syringic acid). Seal with screw cap and shake for 30 seconds. Add 5 mL of methanol/water (8:2, v/v) extraction solution and shake for 1 minute. Extract in a 500W ultrasonic bath at room temperature for 15 minutes. Centrifuge at 5000 rpm for 25 minutes. Take the supernatant phase and filter it through a 0.45 μm filter.

A model 1220 series high performance liquid chromatograph (Agilent, USA) equipped with an Agilent TC-C18 column was used. Mobile phase A is 0.2% phosphoric acid water, and mobile phase B is: methanol and acetonitrile (1:1, v/v). The elution flow rate is 1mL/min. The oven temperature was set to 30°C, and the sample injection volume was 10 μL. The UV detector provides signal at 280nm.

Elution gradient: 0 min, 4% B; 40 min, 50% B; 45 min, 60% B; 60 min, 100% B; 70 min, 100% B; 72 min, 4% B; 82 min, 4% B.

Inject 10 μL of external calibration standard solution (0.030 mg/mL tyrosol solution and 0.015 mg/mL syringic acid solution) and record the chromatogram at 280 nm. Calculate and record the ratio of the response factors of syringic acid to tyrosol, called RRFsyr/tyr.

Inject 10 μL of the sample solution into the HPLC system, and record the chromatogram at 280 nm. The sum of the peak areas of the main biophenolic compounds in the chromatogram (hydroxytyrosol, tyrosol, oleuropein, ligustin and their respective derivative compounds) was compared with the peak area of the internal standard compound syringic acid to calculate the olive Total polyphenol content of oil. Three independent determinations were made on the same sample. Results are expressed as milligrams of compound per kilogram of oil.

Calculated as follows:

RF1μg (syringic acid) = area of syringic acid/number of μg of syringic acid injected

RF1μg (tyrosol) = area of tyrosol/number of μg of tyrosol injected

RRFsyr/tyr=RF1μg (syringic acid)/RF1μg (tyrosol)

ΣA=sum of the peak areas of biophenols (hydroxytyrosol, tyrosol, natural and oxidized oleuropein and ligustin derivatives, flavonoids and phenolic acids) recorded at 280 nm;

As=area of syringic acid internal standard recorded at 280 nm;

W = weight of oil used, g;

Ws = weight of syringic acid used as internal standard in 1 mL of solution added to the sample, mg.

2) Determination of single polyphenol compound content in olive oil:

Weigh 10.0 mg of standard compounds of hydroxytyrosol, luteolin, oleacein, quercetin and other compounds respectively, and dilute to volume in a 10 mL brown volumetric flask to obtain a standard solution of 1.0 mg/mL. Dilute with methanol solution to 1.0 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, 0.2 mg/mL, and 0.1 mg/mL respectively. Record the peak area of each peak at 280 nm using the above HPLC condition method. Make a standard curve of peak area and concentration gradient for each compound. Substitute the peak area corresponding to the chromatogram of the sample solution into the calculated content.

3) Determination of chlorophyll content of olive oil:

Take a 2mL olive oil sample and add n-hexane to dilute it 10 times. The absorbance of the solution was measured using a spectrophotometer and the chlorophyll content of the olive oil was determined colorimetrically. The chlorophyll content has a maximum absorption wavelength at 670nm. 613 is the specific extinction coefficient of chlorophyll. Pigment concentration is calculated as follows:

Chlorophyll (mg/kg)=(A ₆₇₀ ×10 ⁶ )/(613×100×d)

Among them, A is the absorbance and d is the thickness of the spectrophotometer cell (1cm).

4) The antioxidant activity of olive oil was tested using DPPD method and ABTS method respectively.

The test statistical results are shown in the table below and Figures 1 to 3:

As can be seen from the table above, in the polyphenol refined olive oil prepared in Example 1 and Example 4, the total phenolic content is much higher than 250 mg/kg, and the chlorophyll content is lower than 15 mg/kg. Among them, the total phenolic content of the polyphenol refined olive oil prepared in Example 4 is much higher than 250 mg/kg, and it also contains hydroxytyrosol, luteolin, quercetin and Oleacein. The antioxidant activity of the olive oil is greatly improved. , has significant health effects.

In addition, it is worth explaining that:

1) The preparation process of olive leaf polyphenol extract relies on a combination of dark standing treatment, microwave-assisted extraction, and degreening treatment to obtain an olive leaf polyphenol extract with high polyphenol content and removal of chlorophyll. The polyphenol content is significantly increased. .

2) Polyphenol refined olive oil is enriched into conventional refined olive oil by using olive leaf polyphenol extract, preferably using an ultra-high-pressure coupled ultrasonic pulse enrichment process, which has better results.

It is necessary to point out here that the above examples are limited to further elaboration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment, and is not a further limitation of the technical solution of the present invention. used to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. An olive leaf polyphenol extract, characterized in that the preparation method of the olive leaf polyphenol extract includes the following steps: S1, obtain olive leaf raw materials, clean them, and obtain material A; S2, mix material A with water, add acid to adjust the pH to 5.5~6.5, carry out the reaction in the dark and protected from light under stirring conditions, add alkali solution to adjust the pH to 6.5~7.0, let the reaction stand in the dark, vacuum dry, and pulverize. Obtain reaction material B; S3, mix reaction material B with an extraction solvent selected from an aqueous solution containing choline chloride and malic acid, and perform microwave-assisted extraction to obtain extraction liquid C; S4, add acid to the extract C to adjust the pH to 2.5-3.5, mix evenly, add alkali solution dropwise to adjust the pH to 9.5-10.0, let it stand at low temperature to precipitate chlorophyll, and filter to obtain the extract D; S5, mix the extraction solution D with 20% to 55% ethanol aqueous solution, let it stand for layering, and collect the ethanol aqueous phase extract E; S6, distill the ethanol aqueous phase extract E to obtain the ethanol-removed product, and dry it to obtain olive leaf extract powder. 2. The olive leaf polyphenol extract according to claim 1, characterized in that in step S2 and step S4, the acid used is glacial acetic acid, and the alkali solution used is sodium bicarbonate aqueous solution. 3. The olive leaf polyphenol extract according to claim 1, characterized in that the temperature of the dark reaction and the dark reaction are both 35-45°C, and the ambient air humidity is 45%-50%. The duration is 20 to 24 hours. 4. The olive leaf polyphenol extract according to any one of claims 1 to 3, characterized in that the extraction solvent is selected from choline chloride and malic acid containing a mass ratio of 1:2 to 1:4. Aqueous solution, the mass ratio of reaction material B to extraction solvent is 1:10~1:14. The process conditions for microwave-assisted extraction are: microwave power 600~700W, microwave treatment time 15~30min, and treatment temperature 40~50°C. 5. The olive leaf polyphenol extract according to any one of claims 1 to 3, characterized in that the volume ratio of the extract D to the ethanol aqueous solution is 1:1 to 1:4. 6. The olive leaf polyphenol extract according to any one of claims 1 to 3, characterized in that the distillation process parameters are: steam pressure 0.2-2.0Mpa. 7. A polyphenol refined olive oil, characterized in that, in the polyphenol refined olive oil, the total phenolic content is greater than 250 mg/kg, the chlorophyll content is less than 15 mg/kg, and the polyphenol refined olive oil also contains hydroxyl groups Tyrosol, Luteolin, Quercetin and Oleacein. 8. The preparation method of polyphenol refined olive oil according to claim 7, characterized in that it includes the following steps: Obtain the olive leaf polyphenol extract described in any one of claims 1 to 6; The olive leaf polyphenol extract and refined olive oil are evenly mixed according to a mass ratio of 1:15 to 1:40, subjected to high-pressure dissolution-enhancing enrichment and/or pulse ultrasonic dissolution-enhancing enrichment treatment, and centrifugal filtration to obtain a high-content polyphenol extract. Phenolics in refined olive oil. 9. The method for preparing polyphenol refined olive oil according to claim 8, characterized in that the process condition parameters of the high-pressure solubilization treatment are: pressure 150-250MPa, and treatment time 3-10 minutes. 10. The preparation method of polyphenol refined olive oil according to claim 8, characterized in that the process condition parameters of the pulse ultrasonic solubilization treatment are: ultrasonic power is 450~650W, and the total ultrasonic treatment time is 2~10 min. , the ultrasonic pulse interval time program is 10s working, 5s stopping, ultrasonic frequency 250~350Hz, water bath temperature 30~45℃.